Molecular typing of MDR-K. pneumoniae using ERIC-PCR Technique

 

Nihad Shahadha Abed1, Ergin Kariptaş2, Safaa Abed Lateef3

1Department of Advanced Technologies, Faculty of Science Institute, Kirşehir Ahi Evran Universitesi.

2Department of Medical Microbiology, Faculty of Medicine, Samsun University, 55080, Samsun, Türkiye.

3Department of Biotechnology, Faculty of Science, University of Anbar, Anbar, Iraq.

*Corresponding Author E-mail: 0nihad.200@gmail.com

 

ABSTRACT:

Klebsiella pneumoniae is a Gram-negative bacterium that exists naturally in the human body's gastrointestinal tract as part of the natural microflora. It has long been recognized as one of the most frequent nosocomial diseases and a multidrug resistant pathogen. The purpose of the current work was to determine the antimicrobial susceptibility and genetic relatedness of K. pneumoniae strains obtained from Ramadi hospitals, western Iraq, using the ERIC–PCR approach. In the period between June and September 2021, 51 K. pneumonia isolates were obtained from Anbar hospitals. After normal laboratory procedures and differential assays identified K. pneumoniae isolates, disk diffusion susceptibility testing was performed. Also, ERIC-PCR was used to determine genetic relatedness of 15 MDR K. pneumoniae isolates. Kirby –bauer disk diffusion test showed that among K. pneumonia isolates, the highest antibiotic resistance was to Augmentin (96%), cefotaxime (92%), ceftriaxone =Ceftazidime (88%), Amikacin (48%), Levofloxaccin (42%), and imipenem (26%). More than 66.66% of the isolates revealed multi-drug resistant phenotypes. ERIC-PCR results and data analysis indicate that there are 23 distinct ERIC types. The findings of this study show an increase in the prevalence of multiresistant K. pneumoniae in the hospitals studied. The ERIC PCR results revealed a high level of genetic diversity across K. pneumoniae strains, showing that K. pneumoniae isolates are polyclonal in Anbar hospitals.

 

KEYWORDS: Fingerprinting, Multidrug resistance, ERIC – PCR, Molecular typing.

 

 


1. INTRODUCTION:

Klebsiella is a non-motile, Gram-negative, rod-shaped bacterium with a polysaccharide capsule1,2. The capsule covers the entire cells surface and provides resistance against a lot of host defense mechanisms3,4. As an opportunistic pathogen, K. pneumoniae is clinically very important, and 3 to 8% of hospital infections are caused by this species. K. pneumoniae is a prevalent agent in nosocomial infections and causes pneumonia, sepsis, and urinary tract infections, particularly in patients with immunodeficiency5. Infections caused by this bacterium are mainly treated with beta-lactams and fluoroquinolones6,7.

 

Given the importance of infectious diseases threatening public health, especially nosocomial infections in hospitalized patients, understanding the status, distribution, and sources of K. pneumoniae infections can be an important issue; for this purpose, it is proved that molecular typing methods would be helpful. For the epidemiological studies purpose, bacterial molecular typing methods such as ribotyping, pulsed-field electrophoresis (PFGE) and PCR based methods are available8,9. Rep-PCR technique can be noted as an alternative technique to directly produce fingerprints without the use of endonuclease enzymes. In this method, oligonucleotide primers are designed based on sequences of repetitive short repeat nucleotides which are distributed in prokaryotes. This technique is rapid and reproducible and has a high discriminatory power10, 11,12. In this technique, three types of primer sequence are used, which are complementary with three types of repetitive elements including ERIC, BOX, and REP. ERICs are repetitive intergenic sequences of Enterobacteriaceae family with 126 base pairs length 13,14,12. ERIC-PCR fingerprinting methods are widely used for genetic typing. According to recent information about the presence of ERIC sequences in the genome of Escherichia coli k-12, which is fully sequenced and available in Gen Bank, there are about 20 ERIC sequences in its entire genome.

 

Given the importance of K. pneumoniae in nosocomial infections and no report on molecular epidemiology of K. pneumoniae strains in Anbar hospitals, the aim of this study was to examine the antibiotic resistance and genetic relatedness of clinical K. pneumoniae isolates in Borujerd hospitals by ERIC-PCR technique.

 

2. Methodology:

Bacterial strains:

A total of 51 isolates of K. pneumoniae were collected during June to September 2021 from Anbar hospitals. The isolates were identified as K. pneumoniae using conventional microbiological tests , and vitek -2 system 15.

 

Antibiotics susceptibility testing:

Disk diffusion method was used to determine K. pneumonia susceptibility to seven antibiotics including Meropenem, Cefotaxim, Ceftriaxone, Amikacin, Ciprofloxaccin, Augmentin, Ceftazidime based on CLSI 201816 criteria. The antibiotic disks were supplied by Rosco Company, UK.

 

DNA extraction:

Genomic DNAs were extracted from isolates overnight cultures at 37°C using the commercial genomic DNA Purification Kit .

 

ERIC PCR:

Fifteen isolates were selected for analysis by ERIC-PCR based on clinical samples type (urine, sputum, wound, and blood), MDR isolates antibiotic resistance profile, sampling location, hospitals of Anbar. Primer sequence used in this study was: ERIC 1R (5-ATGTAAGCTCC TGGGGATTCA C-3) and ERIC 2 (5-AAGTAAGTGAC TGGGGTGAGCG-3).3μL of DNA template was added to 25μL reaction mixture with 12.5 μL master mix, 2μM of each primer, 5.5 of water (Promega Corporation, USA).

 

PCR program:

Initial denaturation at 95°C for 5 minutes was followed by 35 cycles of denaturation at 94°C for 30 seconds, primer annealing at 47°C for 30 seconds and 52°C for 1 minute, and extension at 72°C for 4 minutes, with a final extension at 72°C for 16 minutes17.

 

ERIC PCR profiles Analysis:

Total Lab software used the Dice method for comparison and the UPGMA method for clustering to detect the strains' similarity based on the analysis of the profiles (Banding).

 

RESULTS:

Collection of samples:

Overall, 62.27% of K. pneumoniae strains were collected from urine samples, 29.41% from sputum, and 5.88% from wounds and 1.96% from blood samples.

 

Susceptibility Test:

Fifty isolates of K. pneumoniae from the wound, burn, sputum and Foley catheter urinary tract infection patients were verified for antibiotic sensitivity by Kirby Bauer disks diffusion method according to the recommendation of18, it`s Susceptibility was tested to seven antimicrobials including Meropenem, Cefotaxim, Ceftriaxone, Amikacin, Ciprofloxaccin, Augmentin, Ceftazidime. The results revealed variation of isolates resistance to all antibiotic that used in the current study. The increase of bacterial resistance towards several antibiotics considered as a tremendous therapeutic difficult.

 

The current study showed that resistance percentage was Augmentin (96%), Cefotaxime (92%), ceftriaxone =Ceftazidime (88%), Amikacin (48%), Levofloxaccin (42%), and Imipenem (26%) (Table 1). According to the results of antibiotic susceptibility testing, a high level of diversity in antibiotic resistance was observed among K. pneumoniae isolates. Resistance to at least one agent of three or more antimicrobial categories were detected in more than 66.66% of the isolates and designated as MDR isolates.

 

Table 1: The antibiogram parameter for study K. pneumoniae isolates according Kirbey-Bauer.

 

Antibiotics

Klebsiella pneumoniae

Resistant

Intermediate

Sensitive

No.

%

No.

%

No.

%

Amox-clav

48

96

1

2

1

2

Ceftriaxon

44

88

3

6

3

6

Cefotaxime

46

92

1

2

3

6

Ceftazidime

44

88

0

0

6

12

Meropenem

13

26

9

18

28

56

Amikacin

24

48

2

4

24

48

Levofoxaccin

21

42

15

30

14

28

*S=sensitive; I=intermediate; R=resistant.

 

Result of ERIC PCR:

Figure 1 shows a gel electrophoresis image of ERIC-PCR products. The number of bands ranged from 4 to 9, and the size ranged from 115 bp to 4008 kb or more. The strains' similarity was determined using the Gel Analyzer's analysis of the profiles (Banding) and the Dice method for comparison and the UPGMA method for clustering. According to the electrophoresis and software analysis results, K. pneumoniae isolates have a high level of genetic diversity (Figure 2).

 

A total of 23 different ERIC profiles (E-types) were observed, two common types include 2 isolates, and one common type includes 3 isolates, and other isolates showed a unique pattern. Indeed, high genetic diversity represents non-clonal distribution of K. pneumoniae strains in studied hospitals. K. pneumoniae strains showed similar antibiotics resistance patterns. K. pneumoniae strains belonging to type (9 and 11 isolate numbers) were isolated from different sources and showed different antibiotics resistance patterns.

 

 

Figure 1: Gel electrophoresis of ERIC products amplification

 

 

Figure 2. Dendrogram of ERIC-PCR results for 15 K. pneumoniae strains isolated from Anbar hospitals.

 

DISCUSSION:

Studies conducted in various countries have shown that Enterobacteriaceae family members are often etiologic agents of UTI, among which K. pneumoniae causes 16-17% of urinary tract infections, and E. coli is the second most common cause of urinary tract infections19,20,21. In this study, 62.27% of K. pneumoniae strains were isolated from urine cultures of patients. In recent years, we have been faced with the emergence of a high level of resistance to available antibiotics in K. pneumoniae worldwide, especially in hospitals. In this study, more than 66.66% of K. pneumoniae strains were multi-resistant. The results of this study and other studies indicated the increase in resistance to antibiotics and the emergence of MDR K. pneumoniae in hospitals22,23. In Iran, studies indicated the genetic diversity in Enterobacteriaceae family members such as E. coli and recently K. pneumoniae by ERIC-PCR24,25. In the present study, a high level of genetic diversity among different strains of K. pneumoniae was observed. In a study by Ramazanzadeh et al. (2013) conducted on the genetic diversity in clinical isolates of E. coli isolated from hospitals of major city in western Iran, ERIC–PCR, allowed typing of the 230 isolates into 205 ERIC types which were then grouped into twenty (C01–C20) as main clusters24,26. Seifi et al. (2016) also reported a high level of genetic diversity among the K. pneumoniae strains in a hospital in Tehran25. Results from studies conducted on other countries also indicated the genetic diversity of K. pneumoniae strains27. In this study, a total of 32 different ERIC profiles were observed among 35 isolates. Thus, 28 isolates of K. pneumoniae were not set in any cluster and formed unique profiles. In fact, the genetic diversity of K. pneumoniae strains represents non-clonal distribution of this bacterium in studied hospitals. All strains collected from urine and showed similar antibiotics resistance patterns.

 

CONCLUSION:

Our findings indicate the suitability and usefulness of ERIC-PCR technique for the purpose of molecular typing and epidemiological studies of nosocomial infections and investigating the genetic diversity among hospital pathogens including K. pneumoniae strains. Our results showed a high level of diversity in antibiotic resistance and ERIC profiles among K. pneumoniae strains isolated from Anbar hospitals. This diversity causes problems for the treatment of infections due to K. pneumoniae strains in hospitals.

 

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Received on 22.10.2021            Modified on 13.01.2022

Accepted on 04.03.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4677-4680.

DOI: 10.52711/0974-360X.2022.00784